DOCSLIB.ORG

(I) I to MY PARENTS for Their Support and Encouragement

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
(I) I to MY PARENTS for Their Support and Encouragement (i) I TO MY PARENTS 4 for their support and encouragement. Y i / (ii) BOTULINUM NEUROTOXINS AND THEIR NEURONAL ACCEPTORS r % by » Richard Stephen Williams Department of Biochemistry Imperial College of Science and Technology * University of London fc A dissertation submitted for the t degree of Doctor of Philosophy of the University of London and the Diploma of Imperial College 4 April, 1984 (iii) # “Poisons can be employed as means for the destruction of life or as agents for the treatment of the sick, but in addition to these two well recognised uses there is a third of particular interest to the physiologist. For him the poison becomes an instrument which dissociates and analyses the most delicate phenomena of living structures and by attending carefully to their mechanism in causing death he can learn indirectly much about the physiological processes of life." * (Claude Bernard) i * ABBREVIATIONS ACh Acetylcho1ine AChE Acetylcholi ne esterase AChR Acetylcholine receptor AMP, ADP and ATP Adenosine mono-, di- and tri-phosphate, respectively BAEE Na-benzoyl-L-arginine ethyl ester bis NN'-Methylenebi sacryl ami de B1WSV Black Widow Spider Venom BoNT Botulinum neurotoxin BrWSV Brown Widow Spider Venom BuTx Bungarotoxin CAM Carboxyami do-methylated CAT Choline acetyl transferase Cl. Clostri di urn CNS Central nervous system CRM Cross-reactive forms ConA Concanavalin A OEAE Diami noethane tetraacetic acid DTT Dithiothreitol DTx Dendrotoxi n EDTA Diaminoethanetetraacetic acid, disodium salt EF-2 Elongation factor-2 EGTA Ethyleneglycol-bi s-(3-ami noethyl ether) N,N'-tetra- acetic acid ELISA Enzyme-1 inked-immunosorbent assay epp End-plate potential GDP and GTP Guanosine di- and tri-phosphate, respectively GERL Go!gi-endoplasmic reticulum-lysosome GTPase Guanosine triphosphatase m GuHCl Guanidinium hydrochloride HPLC High pressure liquid chromatography 125 125I-Bo MT I-labelled botulinum neurotoxin Dissociation constant k d LD50 Lethal dose 50 mepp Miniature end-plate potential MeV Mega electron volt Relative molecular weight M r NAD (P) Nicotinamide dinucleotide (phosphate), oxidised form #* NADH, NADPH Reduced forms of NAD, NADP NAG N-acetylglucosamine NANA N-acetylneuraminic acid % NMJ Neuromuscular junction PAGE Polyacrylamide gel electrophoresis Pi Isoelectric point p l a 2 Phospholipase A2 I PMSF Phenyl methyl sulphonyl fluoride QAE Di ethyl -(2-hydroxypropyl) ami noethyl Q10 Temperature coefficient RCAj Ricinus communis agglutinin (type I) SBA Soyabean agglutinin SDS Sodium dodecyl sulphate % TEMED N,N,N',N'-Tetramethylethylene diamine TLE Trypsin-like enzyme WGA Wheat germ agglutinin (vi) CONTENTS Page Abbreviations ...................................................... (iv) Table of Contents .................................................. (vi) • List of Tables ................................................. ...(xi) List of Figures ..................... (xii) Acknowl edgements ....................... ........................... (xvi) Abstract ................................................................1 * Chapter 1 General Introduction ............................ 3 1.1 Neurotransmi ssi on ................................................ 4 1.1.1 Electrical and chemical synapses................................. 4 * 1.1.2 Modes of neurotransmitter release................................ 8 1.1.2.1 Quantal release of transmitter...........................8 1.1.2.2 Non-quantal release of transmitter....................... 13 £ 1.2 Botulism.........................................................14 1.3 Toxins from Cl. botulinum .................................... 16 1.3.1 Botulinum toxin-haemagglutinin complexes........................16 1.3.2 Structure and activity of the neurotoxins.......................17 * 1.3.3 Bacteriophages and toxigenicity of Cl. botulinum. ............. 20 1.4 The action of botulinum toxins ................................. 21 1.4.1 Interaction with neuronal tissues............................... 21 m 1.4.2 Pharmacological actions of botulinum toxins..................... 23 1.5 Other bacterial and plant toxins ............................... 33 1.5.1 Cholera toxin............................. 33 * 1.5.2 Diphtheria toxin................................................. 34 1.5.3 Tetanus toxin.................................................... 36 1.5.4 Abrin and ricin.................................................. 38 1.6 Presynaptically active snake venom toxins ...................... 40 1.6.1 Snake toxins exhibiting phospholipase A2 activity.............. 40 1.6.2 Venom toxins without PLAg activity.............................. 43 1.7 The present study ...............................................44 (Vii) Page Chapter 2 Purification of neurotoxin from Clostridium botulinum types A and B ............................ 46 2.1 Introduction .................................................... 47 * 2.2 Methods .......................................................... 49 2.2.1 General safety................................................... 49 2.2.2 Purification of neurotoxin from Cl. botulinum type B by affinity and ion-exchange chromatography.................... 50 * 2.2.3 Isolation of type A neurotoxin by affinity and ion- exchange chromatography......................................... 51 2.2.4 Native and SOS gradient-pore gel electrophoresis............... 52 ♦ 2.2.5 Two-dimensional gel electrophoresis..............................53 2.2.6 Ouchterlony double immunodiffusion.............................. 54 2.2.7 QAE-Sephadex chromatography of type A BoNT..................... 54 2.2.8 Other determinations.............................................55 * 2.3 Results ......................................................... 55 2.3.1 Purification of type B BoNT...................................... 55 2.3.1.1 Affinity chromatography of crude toxi n-haemaggl uti ni n compl exes..........................55 2.3.1.2 DEAE-Sephacel chromatography of affinity purified material.............................. 60 * 2.3.2 Purification of type A BoNT...................................... 64 2.3.3 Separation of subunits from BoNT (A) by QAE-Sephadex chromatography...................................................72 ^ 2.4 Discussion ....................................................... 79 Chapter 3 Structural characteristics of BoNT types A and B...85 3.1 Introduction .................................................... 86 (viii) Page 3.2 Methods ................................................ ..91 3.2.1 Preparation of subunits from BoNT (Aand B) by SDS-PAGE......... 91 3.2.2 Amino-acid analysis..............................................92 % 3.2.3 Peptide mapping by limited proteolysis in SDS-acrylamide gels...........................................93 3.2.4 Silver staining of SDS-acryl amide gels.......................... 94 3.2.5 Preparation of subunits from carboxyamidomethylated (CAM)- BoNT (A and B) by high pressure liquid chromatography (HPLC)...95 3.2.6 Peptide mapping of CAM-subunits by reverse-phase HPLC.......... 95 3.2.7 Protein determination in the presence of SDS....................96 * 3.3 Results ..........................................................97 3.3.1 Isolation of subunits from types A and B BoNT by preparative SDS-PAGE.............................................97 k 3.3.2 Amino-acid compositions of subunits from native BoNT (A and B)..................................... 101 3.3.3 Electrophoretic peptide mapping of constituent subunits of types A and B neurotoxins..........................107 * 3.3.4 Rapid preparation of polypeptides from CAM-BoNT by HPLC...... 108 3.3.5 Comparison of reverse-phase HPLC peptide maps obtained from CAM-subunit digests.............................. 113 * 3.4 Discussion ..................... 116 Chapter 4 Radioiodination of types A and B BoNT: their specific interactions with cerebrocortical synaptosomes....................... 123 4.1 Introduction ................................................... 124 4.2 Methods ........................................................ 126 4.2.1 Radiolabelling of botulinum neurotoxins........................ 126 1 oc 4.2.2 Characterisation of I-BoNT (A and B)....................... 127 (ix) Page 4.2.3 Separation of native toxin from its labelled species. ........ 127 4.2.4 Preparation of rat cerebrocortical synaptosomes............... 128 125 4.2.5 Measurement of I-BoNT (A and B) binding to synaptosomes. ..129 * 4.2.6 Effect of temperature and pH on synaptosomal binding of type A 125I-BoNT.................................... 130 4.2.7 Electron-microscope autoradiography of synaptosomes labelled with 125I-BoNT (A).................................... 131 * 4.3 Results ........................................................ 134 4.3.1 Properties of type A *2^I-BoNT................................. 134 4.3.2 Separation of native toxin from itslabelled species........... 138 * 4.3.3 Saturable binding of *2!*i-BoNT (A) to rat cerebrocortical synaptosomes................................... 145 4.3.4 Effect of temperature and pH on synaptosomal ^ binding of type A *^I-BoNT.................................... 153 125 4.3.5 Location of acceptor(s) for type A I-BoNT on synaptic membranes.......................................... 153 4.3.6 Characterisation of acceptor sites for type B * 125 I-BoNT on synaptosomal membranes............................ 157 4.4 Discussion ..................................................... 165 Chapter 5 Nature and selectivity of neuronal receptors for BoNT. ........................................
Load more

Recommended publications
  • Medical Management of Biological Casualties Handbook
    USAMRIID’s MEDICAL MANAGEMENT OF BIOLOGICAL CASUALTIES HANDBOOK Sixth Edition April 2005 U.S. ARMY MEDICAL RESEARCH INSTITUTE OF INFECTIOUS DISEASES FORT DETRICK FREDERICK, MARYLAND Emergency Response Numbers National Response Center: 1-800-424-8802 or (for chem/bio hazards & terrorist events) 1-202-267-2675 National Domestic Preparedness Office: 1-202-324-9025 (for civilian use) Domestic Preparedness Chem/Bio Helpline: 1-410-436-4484 or (Edgewood Ops Center – for military use) DSN 584-4484 USAMRIID’s Emergency Response Line: 1-888-872-7443 CDC'S Emergency Response Line: 1-770-488-7100 Handbook Download Site An Adobe Acrobat Reader (pdf file) version of this handbook can be downloaded from the internet at the following url: http://www.usamriid.army.mil USAMRIID’s MEDICAL MANAGEMENT OF BIOLOGICAL CASUALTIES HANDBOOK Sixth Edition April 2005 Lead Editor Lt Col Jon B. Woods, MC, USAF Contributing Editors CAPT Robert G. Darling, MC, USN LTC Zygmunt F. Dembek, MS, USAR Lt Col Bridget K. Carr, MSC, USAF COL Ted J. Cieslak, MC, USA LCDR James V. Lawler, MC, USN MAJ Anthony C. Littrell, MC, USA LTC Mark G. Kortepeter, MC, USA LTC Nelson W. Rebert, MS, USA LTC Scott A. Stanek, MC, USA COL James W. Martin, MC, USA Comments and suggestions are appreciated and should be addressed to: Operational Medicine Department Attn: MCMR-UIM-O U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) Fort Detrick, Maryland 21702-5011 PREFACE TO THE SIXTH EDITION The Medical Management of Biological Casualties Handbook, which has become affectionately known as the "Blue Book," has been enormously successful - far beyond our expectations.
    [Show full text]
  • Biosafety Manual 2017
    Biosafety Manual 2017 Revised 6/2017 Policy Statement It is the policy of Northern Arizona University (NAU) to provide a safe working environment. The primary responsibility for insuring safe conduct and conditions in the laboratory resides with the principal investigator. The Office of Biological Safety is committed to providing up-to-date information, training, and monitoring to the research and biomedical community concerning the safe conduct of biological, recombinant, and acute toxin research and the handling of biological materials in accordance with all pertinent local, state and federal regulations, guidelines, and laws. To that end, this manual is a resource, to be used in conjunction with the CDC and NIH guidelines, the NAU Select Agent Program, Biosafety in Microbiological and Biomedical Laboratories (BMBL), and other resource materials. Introduction This Biological Safety Manual is intended for use as a guidance document for researchers and clinicians who work with biological materials. It should be used in conjunction with the Laboratory-Specific Safety Manual, which provides more general safety information. These manuals describe policies and procedures that are required for the safe conduct of research at NAU. The NAU Personnel Policy on Safety 5.03 also provides guidance for safety in the workplace. Responsibilities In the academic research/teaching setting, the principal investigator (PI) is responsible for ensuring that all members of the laboratory are familiar with safe research practices. In the clinical laboratory setting, the faculty member who supervises the laboratory is responsible for safety practices. Lab managers, supervisors, technicians and others who provide supervisory roles in laboratories and clinical settings are responsible for overseeing the safety practices in laboratories and reporting any problems, accidents, and spills to the appropriate faculty member.
    [Show full text]
  • Taipoxin Induces Synaptic Vesicle Exocytosis And
    Molecular Pharmacology Fast Forward. Published on February 4, 2005 as DOI: 10.1124/mol.104.005678 Molecular PharmacologyThis article has Fast not beenForward. copyedited Published and formatted. on The February final version 4, may 2005 differ as from doi:10.1124/mol.104.005678 this version. MOL # 5678 Taipoxin induces synaptic vesicle exocytosis and disrupts the interaction of synaptophysin I Downloaded from with VAMP2 molpharm.aspetjournals.org Dario Bonanomi, Maria Pennuto, Michela Rigoni, Ornella Rossetto, Cesare Montecucco and Flavia Valtorta Department of Neuroscience, S. Raffaele Scientific Institute and “Vita-Salute” University, Milan, Italy (D.B., M.P., F.V.); Department of Biomedical Sciences, University of Padova, Italy (M.R., O.R., C.M.) at ASPET Journals on October 1, 2021 1 Copyright 2005 by the American Society for Pharmacology and Experimental Therapeutics. Molecular Pharmacology Fast Forward. Published on February 4, 2005 as DOI: 10.1124/mol.104.005678 This article has not been copyedited and formatted. The final version may differ from this version. MOL # 5678 Running title: Effects of taipoxin on SypI-VAMP2 interactions Address for correspondence: Flavia Valtorta DIBIT 3A3 San Raffaele Scientific Institute via Olgettina 58 20132 Milan, Italy telephone: 39-022643-4826 telefax: 39-022643-4813 e- mail: [email protected] Downloaded from Number of text pages: 29 molpharm.aspetjournals.org Number of tables: = = = Number of figures: 7 Number of references: 33 Number of words in the Abstract: 102 at ASPET Journals on October
    [Show full text]
  • (Oxyuranus) and Brown Snakes (Pseudonaja) Differ in Composition of Toxins Involved in Mammal Poisoning
    bioRxiv preprint doi: https://doi.org/10.1101/378141; this version posted July 26, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Venoms of related mammal-eating species of taipans (Oxyuranus) and brown snakes (Pseudonaja) differ in composition of toxins involved in mammal poisoning Jure Skejic1,2,3*, David L. Steer4, Nathan Dunstan5, Wayne C. Hodgson2 1 Department of Biochemistry and Molecular Biology, BIO21 Institute, University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010, Australia 2 Monash Venom Group, Department of Pharmacology, Monash University, 9 Ancora Imparo Way, Clayton, Victoria 3800, Australia 3 Laboratory of Evolutionary Genetics, Division of Molecular Biology, Ruder Boskovic Institute, Bijenicka 54, 10000 Zagreb, Croatia 4 Monash Biomedical Proteomics Facility, Monash University, 23 Innovation Walk, Clayton, Victoria 3800, Australia 5 Venom Supplies Pty Ltd., Stonewell road, Tanunda, South Australia 5352, Australia * Correspondence: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/378141; this version posted July 26, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Background Differences in venom composition among related snake lineages have often been attributed primarily to diet. Australian elapids belonging to taipans (Oxyuranus) and brown snakes (Pseudonaja) include a few specialist predators as well as generalists that have broader dietary niches and represent a suitable model system to investigate this assumption. Here, shotgun high-resolution mass spectrometry (Q Exactive Orbitrap) was used to compare venom proteome composition of several related mammal-eating species of taipans and brown snakes.
    [Show full text]
  • Environmental Protection Agency § 725.422
    Environmental Protection Agency § 725.422 below in paragraphs (d)(2) through Sequence Source Toxin Name (d)(7) of this section, these comparable Bacillus anthracis Edema factor (Factors I II); toxin sequences, regardless of the orga- Lethal factor (Factors II III) nism from which they are derived, Bacillus cereus Enterotoxin (diarrheagenic must not be included in the introduced toxin, mouse lethal factor) genetic material. Bordetella pertussis Adenylate cyclase (Heat-la- (2) Sequences for protein synthesis in- bile factor); Pertussigen (pertussis toxin, islet acti- hibitor. vating factor, histamine sensitizing factor, Sequence Source Toxin Name lymphocytosis promoting factor) Corynebacterium diphtheriae Diphtheria toxin Clostridium botulinum C2 toxin & C. ulcerans Clostridium difficile Enterotoxin (toxin A) Pseudomonas aeruginosa Exotoxin A Clostridium perfringens Beta-toxin; Delta-toxin Shigella dysenteriae Shigella toxin (Shiga toxin, Shigella dysenteriae type I Escherichia coli & other Heat-labile enterotoxins (LT); toxin, Vero cell toxin) Enterobacteriaceae spp. Heat-stable enterotoxins Abrus precatorius, seeds Abrin (STa, ST1 subtypes ST1a Ricinus communis, seeds Ricin ST1b; also STb, STII) Legionella pneumophila Cytolysin (3) Sequences for neurotoxins. Vibrio cholerae & Vibrio Cholera toxin (choleragen) mimicus Sequence Source Toxin Name (6) Sequences that affect membrane Clostridium botulinum Neurotoxins A, B, C1, D, E, integrity. F, G (Botulinum toxins, botulinal toxins) Sequence Source Toxin Name Clostridium tetani Tetanus toxin
    [Show full text]
  • Snake Presynaptic Neurotoxins with Phospholipase A2 Activity Induce Punctate Swellings of Neurites and Exocytosis of Synaptic Vesicles
    Research Article 3561 Snake presynaptic neurotoxins with phospholipase A2 activity induce punctate swellings of neurites and exocytosis of synaptic vesicles Michela Rigoni1, Giampietro Schiavo2, Anne E. Weston2, Paola Caccin1, Federica Allegrini1, Maria Pennuto3, Flavia Valtorta3, Cesare Montecucco1 and Ornella Rossetto1,* 1Department of Biomedical Sciences and CNR Institute of Neuroscience, University of Padova, Viale G. Colombo 3, 35121 Padova, Italy 2Molecular NeuroPathoBiology Laboratory, Cancer Research UK, London Research Institute, 61 Lincoln’s Inn Fields, London, WC2A 3PX, UK 3Department of Neuroscience, S. Raffaele Scientific Institute and ‘Vita-Salute’ University, Via Olgettina 58, 20132 Milan, Italy *Author for correspondence (e-mail: [email protected]) Accepted 11 March 2004 Journal of Cell Science 117, 3561-3570 Published by The Company of Biologists 2004 doi:10.1242/jcs.01218 Summary The mechanisms of action of four snake presynaptic observed in neurons, but not in astrocytes. These findings phospholipase A2 neurotoxins were investigated in indicate that these snake presynaptic neurotoxins act with cultured neurons isolated from various parts of the rat by same mechanism and that the observed phenotype brain. Strikingly, physiological concentrations of notexin, results from the fusion of synaptic vesicles with the plasma β-bungarotoxin, taipoxin or textilotoxin induced a dose- membrane not balanced by an adequate membrane dependent formation of discrete bulges at various sites of retrieval. These changes closely resemble those occurring neuronal projections. Neuronal bulging was paralleled by at neuromuscular junctions of intoxicated animals and the redistribution of the two synaptic vesicle markers fully qualify these primary neuronal cultures as pertinent synaptophysin I (SypI) and vesicle-attached membrane models for studying the molecular mode of action of these protein 2 (VAMP2) to the bulges, and by the exposure of neurotoxins.
    [Show full text]
  • In-Vitro Neutralization of the Neurotoxicity of Coastal Taipan Venom by Australian Polyvalent Antivenom: the Window of Opportunity
    toxins Article In-Vitro Neutralization of the Neurotoxicity of Coastal Taipan Venom by Australian Polyvalent Antivenom: The Window of Opportunity Umesha Madhushani 1, Geoffrey K. Isbister 2 , Theo Tasoulis 2, Wayne C. Hodgson 3 and Anjana Silva 1,3,* 1 Department of Parasitology, Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka, Mihintale 50300, Sri Lanka; [email protected] 2 Clinical Toxicology Research Group, University of Newcastle, Callaghan 2308, Australia; geoff[email protected] (G.K.I.); [email protected] (T.T.) 3 Monash Venom Group, Department of Pharmacology, Biomedical Discovery Institute, Monash University, Clayton 3800, Australia; [email protected] * Correspondence: [email protected] Received: 15 October 2020; Accepted: 30 October 2020; Published: 31 October 2020 Abstract: Coastal taipan (Oxyuranus scutellatus) envenoming causes life-threatening neuromuscular paralysis in humans. We studied the time period during which antivenom remains effective in preventing and arresting in vitro neuromuscular block caused by taipan venom and taipoxin. Venom showed predominant pre-synaptic neurotoxicity at 3 µg/mL and post-synaptic neurotoxicity at 10 µg/mL. Pre-synaptic neurotoxicity was prevented by addition of Australian polyvalent antivenom before the venom and taipoxin and, reversed when antivenom was added 5 min after venom and taipoxin. Antivenom only partially reversed the neurotoxicity when added 15 min after venom and had no significant effect when added 30 min after venom. In contrast, post-synaptic activity was fully reversed when antivenom was added 30 min after venom. The effect of antivenom on pre-synaptic neuromuscular block was reproduced by washing the bath at similar time intervals for 3 µg/mL, but not for 10 µg/mL.
    [Show full text]
  • Taipoxin.Pdf
    Experimental Neurology 161, 517–526 (2000) doi:10.1006/exnr.1999.7275, available online at http://www.idealibrary.com on The Neurotoxicity of the Venom Phospholipases A2, Notexin and Taipoxin J. B. Harris, B. D. Grubb,1 C. A. Maltin,2 and R. Dixon3 School of Neurosciences and Psychiatry, Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom Received May 17, 1999; accepted September 27, 1999 cular blockade such as anticholinesterases and diamino- The presynaptically active, toxic phospholipases pyridine s (19, 28–30). known as notexin and taipoxin are principal compo- Acute investigations in vivo had suggested that the nents of the venom of the Australian tiger snake and primary cause of death following the inoculation of the the Australian taipan respectively. The inoculation of neurotoxic phospholipases A2 was either a blockade of the toxins into one hind limb of rats caused, within transmitter release or the depletion of transmitter from 1 h, the depletion of transmitter from the motor nerve motor nerve terminals (4, 10, 12). In a more recent terminals of the soleus muscle. This was followed study of the neuropathological consequences of expo- by the degeneration of the motor nerve terminals and sure to toxic phospholipases, Dixon and Harris (7) of the axonal cytoskeleton. By 24 h 70% of muscle inoculated the hind limb of rats with sublethal doses of fibers were completely denervated. Regeneration and b-bungarotoxin (a neurotoxic phospholipase A from functional reinnervation were almost fully restored 2 the venom of the kraits, Bungarus sp.) and showed that by 5 days, but collateral innervation was common in the regenerated muscles, and this abnormality per- the toxin caused the depletion of transmitter from the sisted for at least 9 months.
    [Show full text]
  • (12) Patent Application Publication (10) Pub. No.: US 2014/0005362 A1 Bujko Et Al
    US 2014.0005362A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0005362 A1 Bujko et al. (43) Pub. Date: Jan. 2, 2014 (54) RECOMBINANT CYTOTOXINAS WELL ASA (30) Foreign Application Priority Data METHOD OF PRODUCING IT Dec. 31, 2010 (PL) ..................................... PL393.529 (76) Inventors: Anna Bujko, Kalisz (PL); Magdalena Lukasiak, Parzeczew (PL); Jaroslaw PublicationDCOSSO Classificati Dastych, Lodz (PL); Miroslawa (51) Int. Cl. Skupinska, Poznan (PL); Ewelina C07K I4/2 (2006.01) Rodakowska, Kostrzyn (PL); Leszek (52) U.S. Cl. Rychlewski, Poznan (PL) CPC ...................................... C07K 14/21 (2013.01) USPC ....... S30/371. 530/350, 536/234:435/320.1 (21) Appl. No.: 13/976,914 s s 530370 (22) PCT Filed: Dec. 29, 2011 (57) ABSTRACT The subject of the present invention is a method of modifying (86). PCT No.: PCT/PL2011/050.058 proteinaceous toxins through the addition of an NLS motif. S371 (c)(1), The resulting cytotoxin facilitates the selective elimination of (2), (4) Date: Sep. 16, 2013 proliferating cells, particularly tumour cells. Patent Application Publication Jan. 2, 2014 Sheet 1 of 5 US 2014/0005362 A1 binding translocatin domain domain catalytic domain Drawing 1 Schematic of the structure of PE exotoxin of Pseudomonas aeruginosa. According to: Kreitman (2009), modified. Eissers Drawing 2 Schematic of PE intoxication. Source: Kreitman (2009), Patent Application Publication Jan. 2, 2014 Sheet 2 of 5 US 2014/0005362 A1 pit is rix territats: 8 : EX fissi & 3 & pa208:2678: REEixi: sag opt safe: SSgs; s-r 52s. & S. Aspicin Fig. 1 Map of vector pJ206:26701. Red frames delineate restriction sites for EcoRI and HindIII.
    [Show full text]
  • Search for Relationships Among the Hemolytic, Phospholipolytic, and Neurotoxic Activities of Snake Venoms (Crotoxin Components/Bungarotoxins/Phospholipase A2) T.-W
    Proc. Nati. Acad. Sci. USA Vol. 75, No. 2, pp.'600-604, February 1978 Biochemistry Search for relationships among the hemolytic, phospholipolytic, and neurotoxic activities of snake venoms (crotoxin components/bungarotoxins/phospholipase A2) T.-W. JENG, R. A. HENDON*, AND H. FRAENKEL-CONRATt Department of Molecular Biology and Virus Laboratory, University of California, Berkeley, California 94720 Contributed by H. Fraenkel-Conrat, September 12, 1977 ABSTRACT Several snake venom neurotoxins are larger and deny any direct relationship between the neurotoxicity and the more complex than the well-studied group of postsynaptic toxins hemolytic as well as, presumably, the phospholipolytic activity exemplified by a-bungarotoxin. Several of these, exemplified of this venom. by ,-bungarotoxin, show phospholipase A2 activity (phosphatide 2-acylhydrolase, EC 3.1.1.4) when tested in the presence of de- Snake venom neurotoxins had at that time come to be clas- tergents. The high hemolytic activity of crotoxin, the neurotoxin sified as belonging to either of two groups differing in their site of Crotalus durissus terrificus, in the presence of lecithin has of action. Most of these toxins, exemplified by a-bungarotoxin, been attributed to this activity. The phospholipase A2 activity acted postsynaptically by blocking the acetylcholine receptor of several snake venom proteins has now been compared under sites; a less numerous group, exemplified by f3-bungarotoxin, the physiological conditions of the hemolysis tests. seemed to act presynaptically. The exact mode by which the It appears that only the basic component of crotoxin, B, is enzymatically active, and that its activity is not inhibited by ", type" toxins block neuromuscular impulse transmission re- component A under these conditions, or in the presence of mains uncertain.
    [Show full text]
  • Ibuprofen Targets Neuronal Pentraxins Expresion and Improves Cognitive
    EXPERIMENTAL AND THERAPEUTIC MEDICINE 11: 601-606, 2016 Ibuprofen targets neuronal pentraxins expresion and improves cognitive function in mouse model of AlCl3‑induced neurotoxicity ANUM JAMIL, AAMRA MAHBOOB and TOUQEER AHMED Neurobiology Laboratory, Atta‑ur‑Rahman School of Applied Biosciences, National University of Sciences and Technology, Sector H‑12, Islamabad 44000, Pakistan Received December 30, 2014; Accepted September 1, 2015 DOI: 10.3892/etm.2015.2928 Abstract. Aluminum is known to exert neurotoxic effects Introduction associated with various neurodegenerative disorders, including Alzheimer's disease (AD). Ibuprofen is a well-known Ibuprofen is an over‑the‑counter non‑steroidal anti‑inflam- non‑steroidal anti‑inflammatorydrug, which has demonstrated matory drug (NSAID) (1), which is predominantly used as an potential efficacy in the treatment of numerous inflammatory analgesic, anti‑inflammatory and antipyretic compound (2). and neurodegenerative disorders, including AD. The present Ibuprofen has previously been demonstrated to limit the study aimed to investigate the protective effects of ibuprofen production of pro‑inflammatory cytokines and to exert neuro- on cognitive function, and the expression levels of neuronal protective effects (1). Furthermore, ibuprofen has demonstrated pentraxins (NPs) and interleukin (IL)-1β in an aluminum effectiveness in the treatment of Parkinson's disease (PD) (3), chloride (AlCl3)‑induced mouse model of neurotoxicity. The neuroinflammation associated with β‑amyloid (Aβ) deposi- effects of ibuprofen (100 mg/kg/day for 12 days) on learning tion (4), and motor deficits in hepatic encephalopathy (5), due to and memory were evaluated in the AlCl3-induced neurotoxic its anti‑inflammatory and antioxidant properties. In addition, it mice using a Morris water maze and open field tests.
    [Show full text]
  • Masarykova Univerzita V Brně
    MASARYKOVA UNIVERZITA PEDAGOGICKÁ FAKULTA Katedra fyziky, chemie a odborného vzdělávání Biologické jedy v ţivočišné říši Bakalářská práce Brno 2015 Vedoucí práce: Autor práce: Mgr. Petr Ptáček, Ph.D. Markéta Seborská Prohlášení Prohlašuji, že jsem předloženou bakalářskou práci vypracovala samostatně, s využitím pouze citovaných literárních pramenů, dalších informací a zdrojů v souladu s Disciplinárním řádem pro studenty Pedagogické fakulty Masarykovy univerzity a se zákonem č. 121/2000 Sb., o právu autorském, o právech souvisejících s právem autorským a o změně některých zákonů (autorský zákon), ve znění pozdějších předpisů. …………………………………. V Brně dne 31. března 2015 Markéta Seborská Poděkování Na tomto místě bych ráda poděkovala panu Mgr. Petru Ptáčkovi Ph.D., vedoucímu mé bakalářské práce, za trpělivé vedení a odbornou pomoc mé bakalářské práce. Anotace Bakalářská práce se zaměřuje na toxiny produkované ţivočichy. Věnuje se popisu mechanického účinku toxinů, příznaků intoxikace a terapie otravy. V práci jsou zahrnuty poznatky z toxikologie jako vědního oboru. Jedna kapitola je věnována i historii jedů. Tato práce bude výchozím materiálem pro diplomovou práci. Annotation The bachelor thesis focuses on the toxins produced by animals. It describes the mechanical effect of venoms, symptoms of intoxication and poisoning therapy. This bachelor thesis also includes knowledge of toxicology as a discipline. One chapter is devoted to the history of toxins. This work will be the starting material for a diploma thesis. Klíčová slova Toxikologie,
    [Show full text]